Mitotic recombination occurs in all diploid organisms, but its evolutionary significance has largely been ignored. Mitotic recombination causes loss of heterozygosity (LOH), making it counterintuitive that it could be adaptive. In large multicellular organisms, LOH is considered primarily maladaptive and is frequently associated with tumorigenesis. Yet in organisms with a free-living haploid stage that lack a high genetic load, such as many unicellular fungal and protozoan pathogens, LOH it is likely to be an important mechanism speeding the fixation of beneficial recessive alleles. This assumption is reinforced by studies of natural populations of diploid pathogens that consistently show evidence for polymorphic LOH genomic regions. These observations indicate that LOH is pervasive, however there is a critical need to address whether, under what conditions, and by what mechanisms LOH is an essential component of how diploid pathogens explore their fitness landscapes. Our central hypothesis is that LOH is an important component of evolution by positive selection, but the relative importance of LOH as an adaptive force will be positively correlated with the heterozygosity of the initial genotype or population. We are particularly interested in applying this to the fitness landscape of pathogens because they are notoriously clonal, at least 1,500 described pathogenic protozoon species are diploid, and pathogens appear to have increased rates of LOH in vivo. Here we propose a novel method to test whether heterozygosity speeds the rate of adaptation by mitotic recombination using a yeast-wax worm (Saccharomyces cerevisiae-Galleria mellonella) pathogenesis model. Aim 1 will clonally evolve replicate populations growing inside waxworm larvae initiated from single parental genotypes differing over a 32-fold range of heterozygosity. Using fluorescent cell sorting based on a green fluorescent protein tagged yeast, we will be able to extract pure yeast populations from the infected larvae after 48 hrs of in vivo growth, and this process will be repeated for 100 serial transfers. Cell sorting also allows pathogen fitness to be estimated at each transfer, allowing us to test whether the rate of adaptation is correlated with initial heterozygosity. Aim 2 will use next generation sequencing to genotype the evolved lines to identify parallel LOH events among replicate populations that indicate the action of positive selection and identify virulence genes. Using this experimental evolution approach will allow us to avoid the problems associated genetic drift and mutation accumulation due to small population sizes. Development of the yeast-Galleria infection model into an experimental evolution system will provide a means to map the relationship between pathogen genotype, virulence, and fitness in more powerful way than standard reverse genetic approaches.